Hepatitis C virus (HCV) is a major cause of liver-specific morbidity and mortality worldwide, resulting in >350,000 deaths annually due to cirrhosis and cancer with high rates of disease among those co-infected with human immunodeficiency virus (HIV) or having a history of injection drug use. microRNA-122 (miR-122), an abundant, liver-specific miRNA is essential for replication of infectious HCV and thus plays a novel role in the pathogenesis of chronic hepatitis C. Recent chimpanzee studies have validated miR-122 silencing as an effective antiviral strategy, yet how miR-122 promotes HCV replication is not well understood. Recent work shows that miR-122 promotes viral translation through direct interactions with a miRNA response element at the 5'end of (+)-strand RNA, while new preliminary data indicate that it also acts to stabilize the RNA. This project will investigate the hypothesis that miR-122, in association with Ago and possibly other cellular proteins, forms a ribonucleoprotein complex (the "miRNA-associated stabilization complex", or MASC) at the 5'end of (+)-strand HCV RNA. The MASC complex has several proposed functions: (i) it protects (+)-strand RNA within the cytoplasmic compartment from degradation, thereby increasing RNA available for translation and assembly into membrane-bound replicase complexes;(ii) it may directly enhance the translational activity of HCV RNA;and (iii), it may promote new viral RNA synthesis by facilitating macroassembly of replicase complexes and/or de novo initiation of RNA synthesis. A series of interrelated aims will rigorously test these hypotheses, determine RNA and protein components of the MASC, and further characterize the novel functions of this unique ribonucleoprotein complex. Aim 1 will determine: (a) how miR-122 base-pairs with (+)- strand HCV RNA within the MASC complex;(b) whether RNA stabilization and MASC-increased translation can be functionally uncoupled, and;(c) whether MASC function requires HCV sequences outside the response element. Aim 2 will: (a) combine RNA affinity selection and quantitative proteomics analysis to identify protein components of the MASC complex;(b) validate the presence of these proteins in the MASC complex by IP and assess their role in miR-122 function by RNA interference;(c) develop a cell-free system in which MASC function is recapitulated and the role of host proteins can be further characterized, and;(d) ascertain whether HCV RNA is stabilized by tethering of Ago or other proteins to the 5'RNA. Aim 3 will determine: (a) whether miR-122 regulates macroassembly or stability of replication vesicles by live cell imaging, (b) whether miR-122 is required for negative-strand RNA synthesis, and (c) whether miR-122 facilitates separation of duplexed strands within replicative forms of HCV RNA. Collectively, these studies will provide novel insights into functions of a miRNA that: (a) are important in the pathogenesis of human disease, (b) represent a well-validated but poorly understood therapeutic target, and (c) are unique not only among mammalian viruses but among the Metazoa. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) is a major cause of liver-specific morbidity and mortality worldwide, resulting in >350,000 deaths annually due to cancer and cirrhosis with especially high rates of disease among those co-infected with human immunodeficiency virus (HIV) or having a history of injection drug use. This project will investigate the novel role played by a small cellular ribonucleic acid, miR-122, in this disease. This liver-specific micro-RNA is essential for replication of HCV, and it is a well-validated but very poorly understood target for antiviral therapy. The studies proposed will provide novel insight into how it functions in HCV replication and in the pathogenesis of chronic hepatitis C, thereby enhancing the scientific background on which it can be exploited as a therapeutic target.